We study the mechanisms of the fusion of two biological membranes into one, which is one of the most universal processes in cell physiology. Proteins mediate membrane fusion in exocytosis, intracellular trafficking, and enveloped viral infection, via unknown rearrangements of lipid bilayers. To find out the possible role of membrane lipids in the regulation of these processes we have studied diverse fusion processes including Ca++-triggered cortical exocytosis, GTP-gamma-S -triggered mast cell degranulation and GTP-dependent rat liver microsome - microsome fusion. We have found one class of naturally occurring membrane phospholipids - lysolipids - that acts as a potent inhibitor of membrane fusion in all experimental systems studied. Inhibition was reversible, did not correlate with lysis and could not be attributed to any specific chemical moiety of lysolipids. Fusion was arrested at a stage preceding fusion pore formation. Our results reverse the long-held view that lysolipids promote biologically significant fusion and suggest that highly bent "stalk" intermediates, which favor lipids of the molecular shape opposite to that of lysolipids, are common in disparate fusion processes. Because lysolipids are tightly regulated biological compounds, it is intriguing to speculate that cells control local lysolipid concentrations to regulate fusion processes.